The present application is a national stage application of PCT/DE95/01279, filed on Sep. 15, 1995.
The present invention relates to a process for the production of poly(hydroxyl acids) by means of recombinant bacteria which contain and express at least one fragment of the gene of poly(hydroxy fatty acid) synthase from Thiocapsa pfennigii and which are selected from the group comprising: Pseudomonas putida GPp 104 (pHP1014::E156). Alcaligenes eutrophus PHB 4 (pHP1014::E156), Pseudomonas putida GPp104 (pHP1014::B28+) [DSM #9417] and Alcaligenes eutrophus PHB 4 (pHP1014:B28+) [DSM #9418], whereby the bacteria are cultivated in a mineral medium under aerobic conditions, whereby one offers the bacteria at least one substrate carbon source which is selected from the group consisting of: levulinic acid, salts of levulinic acid, esters of levulinic acid, lactones of levulinic acid, substituted levulinic acid or, as the case may be, its derivatives: 5-hydroxyhexanoic acid, its salts, esters and lactones: 4-hydroxyheptanoic acid, its salts, esters and lactones: 4-hydroxyoctanoic acid, its salts, esters and lactones; their halogenated derivatives us well as their mixtures; one incubates the bacteria for a certain time with the carbon source; and one isolates the poly(hydroxyl fatty acid) polymers that have been synthesized by the bacteria:
a recombinant bacterial strain characterized by the feature that the bacterial strain is selected from the group which comprises Pseudomonas putida GPp104 (pHP1014::B28+) [DSM # 9417] and Alcaligenes eutrophus PHB 4 (pHP1014:B28+) [DSM #9418]:
a poly(hydroxyl fatty acid) produced by any one of the previously described processes:
and a DNA fragment which codes for a pha E component and a pha C component of the poly(hydroxyl fatty acid) synthase from Thiocapsa pfennigii characterized by the feature that it has at least the nucleotide sequence of sequence sections 180 through 1280 (phaE) and 1322 through 2392 (phaC) of the DNA sequence SEQ ID NO:1.
In this age of increasing environmental awareness, there are increasing attempts in industry and science to produce biodegradable polymers. In this regard, these new types of environmentally compatible polymers should essentially have the same properties as those polymers which, for decades, have been prepared via organic chemical synthesis.
In particular in this connection, the ability to process the new types of biodegradable polymers ought to be provided in a similar manner to the processing of conventional plastics using the same methods such as, for example, extrusion, injection molding, injection compression, foaming, etc.
A big disadvantage of organically synthesized plastics is, however, that many of these plastics have enormous biological half-lives or, as the case may be, they cannot be disposed of in garbage dumps or in garbage incineration plants in a non-harmful manner but, rather, aggressive gases are frequently produced such as, for example, in the case of poly(vinyl chloride) which liberates hydrogen chloride gas during incineration.
A first step in the direction of success with environmentally compatible materials was achieved by means of synthetic substances, e.g. the paraffin-like polymers polyethylene and polypropylene since these essentially release only CO2 and water on incineration.
In addition, many attempts have also been made by means of so-called replaceable raw materials such as, e.g. plants that contain a lot of polysaccharide such as potatoes, corn, wheat, beans, peas or similar materials, to obtain the naturally occurring polysaccharides in these plants and to prepare polymers from them which are usable in plastics technology and which are biodegradable.
However, in the case of such polymer materials comprising replaceable raw materials, one is essentially relying on the natural quality of the polymers that occur in these higher plants and only the relatively complex processes of classical cultivation and modern gene technology offer themselves for modification at the genetic level.
An essential further step in the direction of naturally occurring polymers, which are very similar to synthetic thermoplastics, was brought about by the discovery of poly(3-hydroxybutyric acid) by Lemoigne in 1926 [Lemoigne, M. (1926) Products of the dehydration and polymerization of xcex2-oxybutyric acid, Bull. Soc. Chim. Biol. (Paris) 8: 770-782]. The discovery by Lemoigne can be considered to have paved the way for the further development of modern poly(hydroxy fatty acids) which are also designated polyhydroxyalkanoates and represent chemically linear esters of hydroxy fatty acids and hence, ultimately, polyesters.
In the eighties and, especially, in the last five years, further hydroxy fatty acids have been described as components of the poly(hydroxy fatty acids) (PHF) that occur in nature. In this connection, the hydroxyl group of these PHF is usually located in the 3xe2x80x2 position. The aliphatic side chains are either saturated or singly or doubly unsaturated. They are thus non-branched or branched and they can be substituted by functional groups such as, for example, halogen atoms, preferably bromine, iodine and chlorine, or cyano groups, ester groups, carboxyl groups or even cyclic aliphatic groups and even aromatic. In some hydroxy fatty acids, the hydroxyl group is also located in the 4xe2x80x2 or 5xe2x80x2 position.
Poly(hydroxy fatty acids) have been detected previously in gram positive and gram negative groups of bacteria, aerobic and anaerobic groups of bacteria, heterotrophic and autotrophic groups of bacteria, eubacteria and archaebacteria and in anoxygenic and oxygenic photosynthetic groups of bacteria and therefore in virtually all important groups of bacteria. Thus the capability of synthesizing such polyesters apparently does not represent any specially demanding or rare biochemical metabolism. Biosynthesis of the PHF usually sets in when a usable source of carbon is present in excess with the simultaneous deficiency of another nutrient component. In this way, a nitrogen deficiency, a phosphorus deficiency, a sulfur deficiency, an iron deficiency, a potassium deficiency, a magnesium deficiency or an oxygen deficiency can trigger PHF synthesis in bacteria [Anderson, A. J. and Dawes, E. A. (1990) Occurrence, metabolism, metabolic role and industrial uses of bacterial polyhydroxyalkanoates, Microbial. Rev. 54: 450-472; Steinbxc3xcchel, A. (1991) Polyhydroxyalkanoic acids: In: D. Byrom (editor) Biomaterials, Macmillan Press, New York, pages 123-213]. In most bacteria, PHF are deposited in the form of inclusions or grana in cytoplasm, whereby the dry mass of the cell can amount to up to a proportion of 95% by weight.
In eukaryotes, only poly(3-hydroxybutyric acid) has previously been demonstrated as the single PHF. This polyester arises in yeasts such as, for example, Saccharomyces cerevisiae, various plants, e.g. cauliflower, various organs from animals, e.g. the liver and also in humans, e.g. in blood plasma [Reusch, R N. 1992, Biological complexes of polyhydroxybutyrate, FEMS Microbiol. Rev. 103: 119-130]. However, in contradistinction to prokaryotes, the proportion of poly(3-hydroxybutyric acid) in eukaryotes is maximally 0.1% by weight. Inclusions in the form of grana, in the manner in which they occur in prokaryotes, are not known in eukaryotes. As a rule, the eukaryotic PHF are not usually present in free form, either but the polyester is present either linked to other proteins or in the form of a complex which spans the cytoplasm membrane together with calcium ions and polyphosphate molecules.
Thus, only the production of PHF in bacteria is of interest for industrial biotechnological purposes.
The biosynthesis of PHF in bacteria can be subdivided into three phases.
In phase I, the carbon source, which is offered to the bacteria in the medium, is first taken up in the bacterial cells. Either special uptake transportation systems have to exist for the corresponding carbon source or the cells are cultivated under conditions which produce a certain artificial permeability of the cytoplasm membrane with respect to the carbon source. Some non-ionic carbon sources, for example fatty acids in their non-dissociated form, can also get into the cells via passive diffusion.
In phase II, the absorbed carbon source is transformed into a suitable substrate for the particular enzyme which is capable of producing PHF. This enzyme is generally designated poly(hydroxy fatty acid) synthase. Here, numerous more or less complex reaction sequences are conceivable, which can include both anabolic enzymes and catabolic enzymes in the reaction pathway, and these have been demonstrated already, too.
Phase III comprises the linking together of monomeric precursors to give the polyester. This reaction is catalyzed by the enzyme PHF synthase which represents the key enzyme for the biosynthesis of PHF. These enzymes are linked to the PHF grana and they are located there on the surface. Engendered by the very low specificity of most of the PHF syntheses that have previously been examined in this regard and which arise in differing species, the biosynthesis of a plurality of different PHF is possible. Previously, only the co-enzyme A-thioesters of hydroxy fatty acids have been detected in the form of monomeric, bio-synthetically active precursors. As has been shown above, PHF synthase is the key enzyme for PHF synthesis.
After the structure gene of the PHF synthase from Alcaligenes eutrophus had been cloned and synthesized in three different laboratories independently of one another, the structure genes for the key enzyme from approximately 20 different bacteria were cloned [Slater, S. C., Voige, H. and Dennis, D. E. (1988) Cloning and expressing Escherichia coli of the Alcaligenes eutrophus H16 poly xcex2-hydroxybutyrate bio-synthetic pathway, J. Bacteriol. 170: 4431-4436; Schubert, P., Steinbxc3xcchel, A. and Schlegel, H. G. (1988) Cloning of the Alcaligenes eutrophus gene for synthesis of poly-xcex2-hydroxybutyric acid and synthesis of PHB in Escherichia coli, J Bacteriol. 170: 5837-5847; Peoples, O. P. and Sinskey, A. J. (1989) Poly-xcex2-hydroxybutyrate biosynthesis and Alcaligenes eutrophus H16. Identification and characterization of the PHB polymerase gene (phbC), J Biol. Chem. 264: 15298-15303].
At that time, the nucleotide sequences of at least 12 poly(hydroxy fatty acid) synthase genes (PHF synthase genes) were determined. Because of the primary structures of the enzymes, that were derived from this, and because of physiological data, three different types of PHF synthases can now be distinguished. Type I is represented by the PHF synthase from the Alcaligenes eutrophus bacterium which has been examined most thoroughly of all in regard to PHF metabolism and which has a molecular weight of 63,940 and catalyzes the synthesis of PHF from hydroxy fatty acids with a short chain length. In addition to 3-hydroxyvaleric acid and 5-hydroxyvaleric acid are also incorporated into a copolyester comprising different hydroxy fatty acid subunits.
Type II is represented by the PHF synthase from Pseudomonas oleovarans. This enzyme has a similar size to that of the type I PHF synthases (molecular mass 62,400); however, it differs considerably relative to the substrate specificity of the type I PHF synthases. It is capable of incorporating only 3-hydroxybutyric fatty acids of medium chain length into PHF. 4-hydroxy fatty acids and 5-hydroxy fatty acids and 3-hydroxybutyric acid, by contrast, are not incorporated into the bio-synthetic polyesters. However, the specificity of the enzyme is still so broad that approximately 50 different 3-hydroxy fatty acids can be processed as substrates.
Type III is represented by the PHF synthase from Chromatium vinosum. This enzyme resembles the type I PHF synthases from the point of view of substrate specificity. However, it has a distinctly lower molecular mass (approximately 39,730) and needs a second protein in order to be catalytically active.
To the extent that PHF have previously been isolated from bacteria, these do have extremely interesting properties: they are thermoplastically deformable, water-insoluble, biodegradable, non-toxic and optically active provided that they are not homopolyesters of xcfx89-fatty acids. It has also been shown in the case of poly(3-hydroxybutyric acid) that it is bio-compatible and that it has piezoelectric properties.
It has been shown for poly(3-hydroxybutyric acid) [poly(3HB)] and for the copolyester poly(3-hydroxybutyric acid-co-3-hydroxy-valeric acid) [poly(3HB-co-3HV)] that these polymers can be processed with conventional injection molding processes, extrusion blowing processes and injection blowing processes as well as by fiber spinning techniques.
Only two poly(hydroxy fatty acids), namely the homopolyester poly(3HB) and the copolyester poly(3HB-co-3HV), have advanced thus far to large scale production maturity. The copolymer is marketed under the trade name xe2x80x9cBiopolxe2x80x9d.
The production of these biopolymers is disclosed in EP-A 69 497. Production of the polymer is carried out in the form of a two-stage fed-batch process in a 35 m3 air-lift reactor and in tubular kettle reactors with working volumes of up to 200 m3 with a double mutant of Alcaligenes eutrophus as the production organism and with glucose and propionic acid as the carbon sources together with phosphate limitation [Byrom, D. (1990) Industrial production of copolymer from Alcaligenes eutrophus, In: Dawes, E. A. (editor) Novel biodegradable microbial polymers, pages 113-117, Kluwer Academic Publishers, Doordrecht]. The first stage serves for the growth of bacterial cells to high densities and lasts approximately 48 hours, whereby only glucose is offered as the substrate. In the second stage, the cells are grown with phosphate limitation and with glucose and propionic acid as the precursors for the 3-hydroxyvaleric acid component; cell densities of more than 100 g of dry cell mass per liter with a PHF proportion of more than 70% by weight are achieved after a further 40 to 50 hours of cultivation. The cells are then treated with an enzyme cocktail, which essentially comprises lysozyme, proteases and other hydrolytic enzymes, as a result of which the PHF grana are released. The grana sediment on the bottom of the reactor and are collected from there, washed, dried, melted, extruded and granulated.
This PHF is currently produced in a production quantity of approximately 300 metric tons on an annual basis. Although these microbially produced biopolymers, poly(3HB) and poly(3HB-co-3HV), have good properties and can be processed with the methods that are usual in plastics technology, their production is, on the one hand, still very expensive and, on the other hand, the copolymer contains only two monomeric sub-units so that the total properties of the polymer, that is produced, can be controlled only via these two quantities and thus precise control in regard to flexibility, processability in plastics technology plants, resistance to certain solvents, etc. cannot be carried out in fine controlling steps.
Although 3-hydroxyvaleric acid confers good flexibility or, as the case may be, processability on PHF, it has been found that, for example, the component 4-hydroxyvaleric acid, which can additionally be present in the PHF which are synthesized by bacteria, confers on the biopolymer, that is produced, a distinctly higher degree of flexibility than is the case with 3-hydroxyvaleric acid alone.
In the prior art, 4-hydroxyvaleric acid (4HV) has been demonstrated as a new component in bacterial PHF. Various bacteria were capable of synthesizing polyesters with this new component. These are usually copolyesters which also contain 3-hydroxybutyric acid and 3-hydroxyvaleric acid as components in addition to 4HV. However, these terpolymers could previously be produced only starting out from expensive and toxic special chemicals which were offered to the bacteria as precursor substrates or, as the case may be, as a carbon source for PHF biosynthesis.
In particular, Valentin, H. E., Schonebaxc3xcm, A. and Steinbxc3xcchel, A. (1992) Appl. Microbiol. Biotechnol. 36: 507-514 xe2x80x9cIdentification of 4-hydroxyvaleric acid as a constituent of bio-synthetic polyhydroxyalcanoic acids from bacteriaxe2x80x9d describe the manufacture of a terpolyester, which consists of 3-hydroxybutyric acid, 3-hydroxyvaleric acid and 4-hydroxyvaleric acid as subunits, whereby, for example, 4-hydroxyvaleric acid or 4-valerolactone is offered to an Alcaligenes strain as the sole carbon source in a batch process, a fed-batch process or a two step batch process.
The present invention relates to a process for the production of poly (hydroxy fatty acids) as well as recombinant bacterial strains for carrying out the process. In addition, new poly(hydroxy fatty acids) and new substrates for the production of conventional and new poly(hydroxy fatty acids) are described. Moreover, the invention also relates to a DNA fragment, which codes for a PhaE and a PhaC component of the poly(hydroxy fatty acid) synthase from Thiocapsa pfennigii, as well as the corresponding poly (hydroxy fatty acid) synthase.